Effects of shear stress on protein kinase C distribution in endothelial cells

We studied the effects of shear stress on protein kinase C (PKC) in cultured human umbilical vein endothelial cells by use of a flow channel and a monoclonal antibody (MAb 1.3) that recognizes the PKC beta-isozyme. The fluorescence intensity (FI) of the secondary antibody, crystalline tetramethylrhodamine isothiocyanate, was determined by image analysis. The results on each of five shearing experiments were normalized by using the paired stationary control. After 30-min shearing at 2 N/m2, FI per cell increased to 1.6 times that of control, as did the mean FI per unit cell area. The FI per unit stained area and the stained area/cell area ratio were also increased significantly by shearing. The distribution of immunostaining in each cell was determined for its cortical, cytoplasmic, perinuclear, and nuclear regions. The normalized FI per unit area in all four regions and the stained area/cell area ratio in cortical and cytoplasmic regions were significantly higher in the sheared cells than in control; the increases were greatest in the cortical area. Double staining with rhodamine-phalloidin and MAb 1.3 showed the association of actin with the PKC isozyme in both stationary and sheared cells.